EGF secreted from the anchor cell induces multipotent vulval precursor cells (VPCs) to form an invariant 3 deg -3 deg -2 deg -1 deg -2 deg -3 deg pattern. EGF activation of the canonical Ras-Raf-MEK-ERK MAP Kinase cascade induces the most proximal VPC to become a presumptive 1 deg cell, which in turn secretes DSL ligands to induce Notch-dependent 2 deg fate in neighboring VPCs. EGF also contributes to 2 deg fate via an unknown mechanism. To ensure that 1 deg and 2 deg fates are mutually antagonistic, presumptive 2 deg cells express LIP-1/MAP kinase phosphatase to attenuate the Ras-Raf
pro-1 deg signal, while presumptive 1 deg cells degrade
pro-2 deg LIN-12/Notch. Using genetic epistasis analysis we showed that LET-60/Ras functions in parallel with and antagonistic to its Ras-Raf
pro-1 deg function. In addition to Raf, Ras signals through RalGEF, a non-canonical Ras effector recently shown to be critical in several Ras-dependent cancers. The principal function of RalGEF is to activate the small GTPase Ral. As we found with LET-60/Ras, the C. elegans RalGEF and Ral orthologs RGL-1 and RAL-1 antagonize Ras-Raf
pro-1 deg signaling, suggesting that the Ras-RalGEF-Ral signaling module is evolutionarily conserved and counteracts Ras-Raf
pro-1 deg activity. Mutationally activated RAL-1 induced 2 deg fate in a weakly constitutive LIN-12/Notch background. Therefore, we used mutationally activated LET-60/Ras harboring a RalGEF-selective effector binding domain mutation (E37G) to similarly promote Notch-sensitized 2 deg fate, and this activity was RGL-1- and RAL-1-dependent. Likewise, endogenous mutant activated LET-60/Ras stimulated RGL-1- and RAL-1-dependent 2 deg fate when LIN-45/Raf was abrogated and LIN-12/Notch was sensitized. Therefore, Ras-RalGEF-Ral signaling is
pro-2 deg , explaining its antagonism of Ras-Raf
pro-1 deg signaling. During induction GFP expression driven by the
ral-1 promoter was restricted to presumptive 2 deg cells, suggesting that transcriptional exclusion of RAL-1 from the presumptive 1 deg cell provides a mechanism for LET-60/Ras effector switching from Raf to RalGEF-Ral. Thus, we have shown that Ras switches effectors as part of a developmental patterning program. Previous studies showed that strong EGF signaling induced 1 deg fate, while weaker EGF signaling induced 2 deg fate. We titrated the EGF (LIN-3) or EGF receptor (LET-23) signal to levels sufficient to promote 2 deg but not 1 deg fate, and found that
pro-2 deg EGF signal was LET-60-, RGL-1- and RAL-1-dependent, implying that the
pro-2 deg EGF signal is propagated through Ras activation of RalGEF-Ral signaling. We have therefore identified the long-missing mechanism by which EGF promotes 2 deg fate.